Team:UC Chile/Cyanolux/Results short

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<h2>Plasmids backbones</h2>
<h2>Plasmids backbones</h2>
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We built two integrative plasmid backbones for Synechocystis PCC. 6803.
 
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[[File:UC_Chile-IntKplasmidResult.jpg| 400px | left]]
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We built two integrative plasmid backbones for Synechocystis PCC. 6803 (see design [https://2012.igem.org/Team:UC_Chile/Cyanolux/Project_short#Wetlab_strategy here])
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[[File:UC_Chile-IntSplasmidResult.jpg| 560px | right]]
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[[File:UC_Chile-IntSplasmidResult.jpg| 560px | right]]
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[[File:UC_Chile-IntKplasmidResult.jpg| 400px | left]]
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<h2>Constructs for Synechocystis</h2>
 
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Using our plasmid backbones we proceeded to build our constructs for <i>Synechocystis</i> (LuxAB & LuxCDEG)
 
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[[File:UC_Chile-IntKfullplasmidResult.jpg| 480px | left]]
 
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[[File:UC_Chile-IntSfullplasmidResult.jpg| 480px | right]]
 
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Plasmids were verified by digestion and then corroborated by sequencing.
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<h2>Constructs for Synechocystis</h2>
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Using our plasmid backbones as a starting point, we proceeded to build our constructs for <i>Synechocystis</i>:
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[[File:UC_Chile-IntKfullplasmidResult.jpg| 640px]]
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Even though we tried many times (>6) to assemble the substrate production (LuxCDEG) plasmid,we were unable to obtain a correct product. We are currently designing another strategy to build the construct...
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<h1>Characterization in Synechocystis</h1>
<h1>Characterization in Synechocystis</h1>
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<h2>Transformation</h2>
<h2>Transformation</h2>
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We used our plasmid backbones to transform Synechocystis PCC. 6803.
 
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-Image of Syne plates with colonies-
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We used our plasmid backbones to transform Synechocystis PCC. 6803 to verify that our initial design could indeed integrate into <i>Synechocystis's</i> genome.
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Transformation with pSB1C3_IntK (BBa_K743015):
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[[File:Pta_LuxABvf.JPG| 600px| center]]
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Transformation with pSB1C3_IntS (BBa_K743010):
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[[File:Trans_IntS.JPG| 600px| center]]
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After two weeks, colonies became apparent in the transformation plates for both plasmid backbones while in negative controls (transformations with no DNA) there was complete absence of surviving colonies.
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<h2>Verification of constructs</h2>
<h2>Verification of constructs</h2>
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We proceeded to verificate the integration of the constructs in Synechocystis.
 
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(New image with illustration of verification from outer RS1 and RS2 - October 1X something)
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We proceeded to verify the integration of the constructs in <i>Synechocystis</i> by doing multiple PCRs to amplify various parts.
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[[File: UC_Chile-verification.jpg]]
<h2>Bioluminescence Assays</h2>
<h2>Bioluminescence Assays</h2>
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We proceeded to check if our <i>Synechocystis</i> strain with the Luciferase genes did indeed produce light in the prescence of exogenous substrate.
<h3>Bioluminometer</h3>
<h3>Bioluminometer</h3>
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We measured bioluminescence by adding directly the substrate to the cells and measuring light-output in a Luminometer.
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<html><center><img src="https://static.igem.org/mediawiki/2012/6/6a/Kjasfkjhasdf.jpg" align="right" width="600"></center></html>
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While we could measure bioluminescence of the positive LuxBrick E.coli controls, no apparent bioluminescence was seen in our <i>Synechocystis</i> cells. This led us to think that the problem might be the size of the promoter, which if not long enough, would not be able to recruit necessary transcription factors for expression.
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<h3>High-sensitive camera</h3>
<h3>High-sensitive camera</h3>
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During the Latin America Jamboree, we had a chat with a couple of judges and a student from Universidad de los Andes, David Olarte, on how to induce Synechocystis with n-decanal.
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David sent us some work he had done on luminescence assays in cyanobacteria. Following his methods we finally were able to see light emission, confirming presence of catalytically active luciferase. Thanks David!
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<html><center><img src="https://static.igem.org/mediawiki/2012/7/7f/Cam.results.finalisimo.jpg" align="right" width="600"></center></html>
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<h1>Experimental Highlights</h1>
<h1>Experimental Highlights</h1>
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<div style="float: right">
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[https://2012.igem.org/Team:UC_Chile/Cyanolux/Results See more]
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- Constructed two functional integration plasmids for <i>Synechocystis</i> PCC. 6803
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- Transformed <i>Synechocystis</i> PCC. 6803 with an optimized transformation protocol, available [https://2012.igem.org/Team:UC_Chile/Protocols#Transformation_of_Synechocystis_PCC._6803 here]
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- Verified integration of constructs
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- Confirmed expression of genes and activity of luciferase through a bioluminescence assay with exogenous substrate.
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<a href="https://2012.igem.org/Team:UC_Chile/Cyanolux/Modelling"><img src="https://static.igem.org/mediawiki/2012/a/ab/UC_Chile-Continue_button.jpg" align="right">
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Latest revision as of 04:01, 27 October 2012

Project: Luxilla - Pontificia Universidad Católica de Chile, iGEM 2012